This proposed project is in response to the need for more extensive toxicological evaluation of environmental chemicals and for better predictive models to assess associated risks of chemical exposure to humans. There is also need for innovative methods for evaluating the effects of chemicals on pluripotent stem cells and the differentiation process. Maintenance of stemness and the stem cell differentiation processes is regulated by networks of genes controlled, at least in part, by the stem cell epigenome. Disruption of this finely tuned regulatory circuit by exposure to certain environmental agents can lead to adverse health effects. Chromatin in pluripotent stem cells is characterized by several unique properties, including a novel structure highly enriched in embryonic stem (ES) cells in which nucleosomes bear juxtaposed repressive and activating histone modifications (bivalent domains) that direct gene expression. Bivalently marked histones silence developmental genes in ES cells while keeping them primed for activation upon initiation of specific differentiation programs. The unique characteristics of chromatin and poised status of bivalently marked genes in stem cells may render them particularly sensitive targets for epigenetic effects resulting from chemical exposure. To expand the biological landscape of toxicological evaluation of chemicals, the objective of this project is to develop a medium throughput assay platform to monitor pertinent histone modifications at a panel of developmentally-relevant genes in human embryonic stem (hES) cells. A comprehensive toxicogenomics evaluation of gene expression changes will be performed using a training set of chemicals representing various classes of chemicals known to affect enzymes that modulate histone acetylation and methylation, including some known teratogens. Correlating changes in specific active and repressive histone marks at the promoters of consensus differentially expressed genes will then be identified to define a biomarker signature indicative of an epigenetic response in stem cells. The signature will be validated by blind testing of a set of chemicals using a ChIP assay platform that allows for all immunoprecipitation steps to be performed in a single 96-well plate. The following specific aims are proposed to accomplish these goals: 1) conduct expression profiling and ChIP evaluation of hES cells exposed to chemicals known to influence histone acetylation/methylation using a training set of ?40 compounds to construct a predictive transcriptome-based signature of epigenetic impact on developmental processes; 2) establish methodology for preparing sheared chromatin from hES cells directly in the chemical exposure plate and for conducting ChIP in a 96-well assay plate; 3) develop and implement bioassay standard procedures and quality control criteria; and 4) test the epigenetics-based signature derived from the training set of chemicals with a blinded set of test chemicals. This project will result in a medium throughput platform for rapid and efficient screening of effects of environmental toxicants on the human epigenome that could lead to developmental defects or predispose an individual to disease.